Fig. 1

A new BRAF fusion BAIAP2L1-BRAF was detected in a PTC patient. A New BAIAP2L1-BRAF fusion revealed in patient 17 by RNAseq analysis. B Venn diagram and C. Volcano plot representing the differential expression of proteins detected in the mass spectrometric data from normal tissues compared to the corresponding tumor lesions of 4 patients (#14, #15, #17, #21) (Benjamini-Hochberg corrected t-test, p > 0.01, log2(fold change) > 0.4, detected in at least 9 LC-MS runs). D Western blot detection of TRIM25, PKCδ and IQGAP1 in normal and tumor/metastasis tissues of several patients of an additional cohort. E Relative quantification of PKCδ n = 11), TRIM25 (n = 15) and IQGAP1 (n = 10) protein levels obtained from Western Blot analysis of various patients’ protein extracts (Tumor and Metastasis vs Normal) (error bars = SEM, paired t test, two-tailed, P value: ** < 0.01). GAPDH was used for normalization. F MAPK pathway activation detected by Western blotting of proteins extracted from Nthy-ori 3–1 cells transiently transfected with EV and BAIAP2L1-BRAF WT. G 3D MTT of Nthy cells stably expressing EV, BAIAP2L1-BRAF-FLAG WT or different BAIAP2L1-BRAF mutants after colony formation assay (error bars = SEM, n = 5, n = 4 for R188L, Dunnett’s multiple comparisons test, P value: * < 0.033, **** < 0.0001, ns-not significant). H Fluorescence microscopy images of Hoechst (blue)/phalloidin (red) staining of spheroids from cells stably expressing EV and BAIAP2L1-BRAF-FLAG WT 3 days after seeding (left) or embedded in Matrigel for 72 h (right). Scale bar: 200 μm. I Same as in G, but quantification of the spheroid diameters. Spheroids of Nthy cells stably transfected with EV, BAIAP2L1-BRAF-FLAG WT or BAIAP2L1-BRAF-FLAG mutants were analyzed (error bars = SEM, n = 6, Dunnett’s multiple comparisons test, P value: **** < 0.0001, ns-not significant). J Kinase activity assay of Nthy-ori 3–1 cells transiently transfected with EV, BAIAP2L1-BRAF WT, kinase-dead (KD) or coiled-coil domain deleted fusion (ΔCC) expressing plasmids. K Western blot detection of a crosslinking and release experiment using DTME and DTME+DTT, respectively. Nthy-ori 3–1 cells were transiently transfected with BAIAP2L1-BRAF-FLAG WT plasmid and treated for 1 hour with DTME or DMSO and for 15 minutes with DTT. L MAPK pathway detected by Western blotting of proteins extracted from Nthy-ori 3–1 cells of cells stably expressing BAIAP2L1-BRAF-FLAG WT, treated for 1 hour with DMSO, vemurafenib, trametinib or PLX-8394 with indicated doses. Representative Western blots are shown